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Effects of phenobarbital administration on the histology of the liver and

brain, and the activities of some biochemical parameters of the liver of Wister
rats

Article · August 2009

DOI: 10.4314/br.v7i1.45470

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Bio-Research, 7(1): 446 – 450 446

Effects of Phenobarbital Administration on the Histology of the Liver and

Brain, and the Activities of Some Biochemical Parameters of the Liver of
Wister Rats
1
Eze, A. A., 2Nwachukwu, D. C., 3Achukwu, P. U. and 3Okwuosa, C. N.
1
Department of Medical Biochemistry, University of Nigeria, Enugu Campus, Enugu, Nigeria.
2
Department of Physiology, University of Nigeria, Enugu Campus, Enugu, Nigeria.
3
Department of Medical Laboratory Sciences, University of Nigeria, Enugu Campus, Enugu, Nigeria.

Corresponding Author: Eze, A. A. Department of Medical Biochemistry, University of Nigeria, Enugu

Campus, Nigeria. Email: toniuseze2005@yahoo.com Phone: +234 8064945917.

Abstract

The effects of phenobarbital on the histology of the liver and the brain and on selected biochemical
parameters of the liver of wistar rats were studied. Histological examination showed prominent
lesions in the liver and brain of the tested groups of rats. Biochemical analysis revealed significant
(P < 0.05) increase in the activities of alkaline phosphatase, alanine transaminase and in the level of
cholesterol, with increased drug dosage. These correlated with the pathological changes observed
in the liver and the brain of the wistar rats. The effects of phenobarbital on the liver and brain cells
were found to be dose-dependent. Half the LD50 of the drug (8mg/160g) administered on the rats
caused reasonable injuries to the tissues of the liver and the brain of the wistar rats.

Keywords: Phenobarbital, Liver, Brain, Alkaline Phosphatase, Alanine Transaminase, Cholesterol.

Introduction reticulum, and induction or repression of

There has been a resurgence of clinical interest
in the role of phenobarbital (PB) for treating
childhood epilepsy in Western countries (Pal,
2007). In 1912, PB became one of the first
agents used against epilepsy (Shorvon and
Farmer, 1988), and is today the most widely used
anti-epileptic drug (AED) in the world (Pal, 2007).
In resource-poor countries, an anticonvulsant that
controls seizures and prevents recurrence would
be ideal (Kokwaro et al., 2003). Diazepam is
useful in status epilepticus, but it does not offer
prophylaxis following single-dose administration
(Ogutu et al., 2002), whereas multiple doses can
cause respiratory depression (Crawley et al.,
2000). Phenobarbital would be ideal for use in
resource-poor countries, since it is cheap, readily
available, fast-acting and can be administered
i.m. at peripheral health facilities with few
resources (Kokwaro et al., 2003). In western
countries, phenobarbital has been in and out of
favour as an AED (Pal, 2007). Although widely
used as a prophylactic against febrile seizures in
the 1960s and 1970s, concerns about its
neurobehavioral adverse effect profile led to a
decline in its use for all seizure disorders
(Shorvon, 1986).
The reasons for renewed interest in
phenobarbital arise mainly from doubts about the
safety of new licensed pharmaceuticals and
public disquiet over the unsatisfactory regulatory
framework for preclinical evaluation of new
medicines, particularly for children (Pal, 2007).
Cost of treatment may also be a consideration:
carbamazipine costs around ten times, and
lamotrigine almost forty times as much as the
equivalent dose of phenobarbital (Pal, 2003).
The effects of phenobarbital on liver
physiology are typified by hepatic hypertrophy,
hyper proliferation of the smooth endoplasmic
numerous genes, especially the genes of
cytochrome P450 enzymes (Garcia-Allan et al.,
2000). Hepatic hypertrophy induced by
phenobarbital is mediated by a moderate
increase in hepatocyte DNA synthesis and
dramatic enlargement of individual hepatocytes
(Carthew et al., 1998). Hepatic enlargement
subsides after phenobarbital withdrawal, and this
decrease in liver size is mediated by hepatocyte
apoptosis (Bursch et al., 1984). Also, liver
regeneration in response to partial hepatectomy
is markedly diminished in phenobarbital–treated
rat and mouse livers (Aletti et al., 1981).
Phenobarbital is also the prototype of liver tumor
promoters, dramatically increasing tumor
numbers when chronically administered after
initial genotoxic carcinogen treatment (Bell and
Michalopoulos, 2006).
However, phenobarbital is still being
used as a sedative in cases of gastrointestinal
and asthmatic functional disorders, as well as to
antagonize the adverse central stimulant effects
of some drugs such as ephedrine,
dextroamphetamine or theophylline. It is also
used in cases of withdrawal syndromes of
hypnosedative agents (Lopez-Munoz et al. 2005).
In the field of neurology, phenobarbital is still
employed, not only in the treatment of certain
types of epilepsy (partial and tonic-clonic
generalized seizures), but also in the emergency
treatment of some types of convulsions, such as
those associated with tetanus, eclampsia,
cerebral hemorrhage, status epilepticus, or
different forms of poisoning (Lopez-Munoz et al.,
2005). Phenobarbital is also capable of
improving the hepatic transport of bilirubin in
patients with hemolytic jaundice, so that it can be
used in newborn babies to treat
hyperbilirubinemia and kernicterus (Lopez-Munoz
et al., 2005).

Bio-Research Published June 2009 ISSN 1596-7409

Eze et al. 447

The present study is therefore aimed at of distortion from the normal cell framework with
determining the effects of phenobarbital on the the greatest level being observed in test group 4
liver and brain of rats, with a view to determining whose animals received the largest drug dose
whether it can cause any histological alteration(s) (Figs. 1 - 6).
in these organs. The effects of phenobarbital
dosage and duration of administration on
selected biochemical parameters of the liver were
also studied.

Materials and Methods

Drug administration: The LD50 of phenobarbital

sodium (PHTNa) in rats is 100mg/kg (Ruch et al.,
2003). Administration was intraperitoneally, the Fig. 1: The photomicrograph of
starting does being about one-third of the LD50. an apparently normal liver tissue
The test groups (groups 1-4, below) received of rat (x 200), showing normal
respectively, a corresponding increase in drug hepatocytes (h); clear and normal
dose while the control group (5) received no central canal (c).
administration of drug. Drug was administered
daily for a period of 3 weeks (Table 1).

Table 1: Dosage of phenobarbital sodium

administration
Groups Dose mg/160g Drug in mg/kg ml/l
body wt
1 (6 rats) 5.0 31.25 0.25
2 (6 rats) 6.0 37.50 0.30
3 (6 rats) 7.0 43.75 0.35
4 (6 rats) 8.0 50.00 0.40
5 (6 rats) 0.0 - -
Fig. 2: The photomicrograph of liver
Animal sacrifice: Ten rats, comprising of two tissue of rat (x200) administered with
rats from each group, were randomly selected 5mg/160g (phenobarbital/body weight),
and painlessly sacrificed at the end of every week after 3 weeks of administration, showing
of drug administration by placing them in a normal hepatocytes (h); frank red blood
cells (f) in central canal; mild vacuolation
container having chloroform-soaked wool. Their (v).
post mortem was performed immediately and the
organs for study resected and fixed promptly in Observations made from the liver
10% formal saline. Subsequently they were microphotographs showed that progressive
histological processed, their sera were collected increase in period of administration of
and analyzed biochemically at each sacrifice for phenobarbital initiated necrosis in the liver of the
alanine transaminase, alkaline phosphatase and rats (Fig. 2). The degenerative processes
cholesterol levels (Mayne, 1994). intensified evidenced by a large increase in
vacuolation, as the dosage of phenobarbital was
Tissue processing: The liver and brain excised increased (Fig. 2).
from the dissected rats were cleared of the
adhering connective tissue, fixed for 24 hours by
immersion in equal parts of 10% formal buffered
saline. There after, fixed tissues were
dehydrated in ascending grades of ethanol,
cleared in xylene and embedded in liquid paraffin
wax. The tissues were sectioned to a diameter of
3-5 microns using the Heitz 150 rotary microtome
(Cambridge model). The sections were then
subjected to Erlich’s Haematoxylin and Eosin (H
and E) staining technique as described by Baker
and Silverton (1985). Sections were examined
using swift binocular microscope with in-built
lighting system and were photographed.
Fig. 3: Photomicrograph of the normal
Results cerebellum of the control group of rats
(x200), showing the normal distribution
Histopathology: On examination of the of glial cells: macrocytes (a) in brain
photomicrographs from the five different groups cortex (c); microcytes (i) in brain medulla
of experimental animals, the control showed (m).
normal architecture of the tissues under study.
The other four groups presented different levels
Effects of phenobarbital administration on organ histology and biochemical activities of rats 448

Biochemical parameters: The activities of two

liver enzymes, alanine transaminase and alkaline
phosphatase, together with cholesterol level in
the serum of rats were analyzed against drug
dose and duration of administration. The effect of
the drug was observed to be significantly dose
dependent (p < 0.05). Using a one-way analysis
of variance, the increase in biochemical
parameters within the three weeks of drug
administration was found to be significant (p <
0.05).

Table 2: Cholesterol levels during the period

of drug administration
Fig. 4: Photomicrograph of brain tissue Dose of Cholesterol Cholesterol Cholesterol
cerebellum of rats (x200) from test group 1, pheno- levels levels in wk 3
administered with 5mg/160g (phenobarbital barbital in wk 1 in wk 2 (mmol/l)
/ body weight) for 3 weeks. Contracted (mg) (mmol/l) (mmol/l)
microglial cells adjusting to the initial 0.0(control) 2.8 3.0 3.0
effects of phenobarbital; macrocytes (m). 5.0 3.1 3.2 3.6
6.0 3.5 3.9 4.1
7.0 4.3 5.0 5.8
8.0 6.1 7.0 7.6

Table 3: Alanine transaminase (ALT) levels

during the period of drug administration
Dose of ALT ALT ALT
pheno-barbital levels levels levels
(mg) in wk 1 in wk 2 in wk 3
Fig. 5: Photomicrograph of brain (iu/l) (iu/l) (iu/l)
tissue cerebellum of rats (x200) from 0.0(control) 10 11 11
test group 2, administered with 6mg - 5.0 12 12 13
7mg phenobarbital /160g body weight 6.0 12 13 13
for 1 week. Here, there is a reduction 7.0 13 12 13
in microglial cells (i); macrocytes (a) 8.0 19 21 25
are fairly constant
Table 4: Alkaline phosphatase (ALP) levels
during the period drug administration
Dose of ALP ALP ALP
pheno-barbital levels levels levels
(mg) in wk 1 in wk 2 in wk 3
(iu/l) (iu/l) (iu/l)
0.0(control) 33 34 33
5.0 69 76 87
6.0 73 78 82
7.0 88 86 98
8.0 90 95 109

Tables 2, 3 and 4 show the changes in the levels

Fig. 6: Photomicrograph of brain tissue
cerebellum of rats (x200) from test group,
administered with 8mg/160g (phenobarbital
/ body weight) for 2 weeks. Here, there are
cloudy, crowded neuronal cells; there are
large pink-stained glial cells. The onset of
liquefaction is visible.
Figure 3 was obtained from the control group of
the rats, and showed normal brain tissue
architecture. Figures 4-6 however, displayed
neurons with pyknotic nuclei and necrotic cells.
Here, brain cells were largely replaced by pink-
staining debris, with phagocytic cells (microcytes)
engulfing degenerate materials. Figure 6
represents brain tissues of group 4 rats, which
were administered with 8mg of the drug per 160g
body weight. They exhibit an extensive
neurodegeneration with an onset of gliosis.
of cholesterol, alanine transaminase and alkaline
phosphatase respectively, with increase in
phenobarbital dosage. Table 2 shows a
progressive increase in cholesterol level with
increase in the period of phenobarbital
administration, for each test group. There was
also an increase in cholesterol level with increase
in phenobarbital concentration. Similarly, the
levels of both alanine transaminase (table 3) and
alkaline phosphatase (table 4) increased with
increase in the period of phenobarbital
administration, and with increase in phenobarbital
concentration.
Using a one-way analysis of variance
(ANOVA), the level of significance (p<0.05) of
these increases with increased administration of
phenobarbital was determined (table 5). It was
found that increase in the level of either
cholesterol, alanine transaminase or alkaline
phosphatase was significant (p<0.05).
Eze et al. 449

Table 5: One-way analysis of variance for the determination of the level of significance of increases
in the biochemical parameters with dosage of phenobarbital
Parameters Sum of df Mean square F P
squares
Cholesterol level Between groups 29.600 4 7.400 0.05
(mmol/l) in weeks Within groups 2.773 10
Total 32.373 14 0.277 26.683
Alkaline Between groups 7566.933 4 1891.733 39.193 0.05
phosphatase level Within groups 482.667 10
(iv/l) in weeks Total 8049.600 14 48.267
Alanine Between groups 228.667 4 57.167 26.797 0.05
transaminase (iv/l)
in weeks 2.133

Discussion administration. Exp. Mol. Pathol., 34:

A rise in plasma activities of alanine
transaminase has been shown to characterize
liver-cell damage, while alkaline phosphatase
activities are increased in cholestasis (Mayne,
1994). The results obtained from this study
showed significant increases (P < 0.05) in the
plasma activities of alkaline phosphatase and
alanine transaminase. Plasma level of cholesterol
also increased significantly (P < 0.05). This
showed that high doses of phenobarbital or
discrete doses administered for long periods had
adverse effects on the liver. This finding is further
supported by the histological results which
showed the formation of cellular vacuoles on
administration of very mild doses of
phenobarbital.
The histological results obtained from
the brain tissue showed that phenobarbital
causes damage to the brain. Neurodegeneration
increases as phenobarbital dosage are increased
progressing ultimately to gliosis and then
liquefaction. This is in support of the finding by
Yazar et al., (2002) that phenobarbital increased
the activities of marker enzymes in the brain and
the liver of mice. It has also been shown that
phenobarbital increased the level of serotonin in
the forebrain and cerebellum of mice with a
hereditary susceptibility to seizures (Matsumoto
et al., 1983). Similarly, Lewin and Bleck (1997)
found that phenobarbital inhibits seizures by
decreasing the activities of neurons. These
findings agree considerably with the histological
results obtained in this study which showed
features that can indeed impair transmission of
impulses and hence the functional ability of the
brain.
From all indications however,
phenobarbital will continue to be clinically
relevant in the treatment of epilepsy and other
types of convulsions owing to the fact that it is
very cheap compared to other remedies. It is
therefore necessary to advice that the drug be
taken only on the prescription and under the strict
supervision of a qualified medical personnel.
References
Aletti, M. G., Presta, M. and Ragnotti, G. (1981).
Liver growth after partial hepatectomy:
influence of Phenobarbital
216-225.
Baker, F. J. and Silverton, R. E. (1985).
Introduction to Medical Laboratory
th
Technology, 17 ed., Butterworth-
Heinemann, Oxford.
Bell, A. W. and Michalopoulos, G. K. (2006).
Phenobarbital Regulates Nuclear
Expression of HNF -4α in Mouse and
Rat Hepatocytes Independent of CAR
and PXR. Hepatology, 44: 186-194.
Bursch, W., Lauer, B., Timmermann-Trosiener, I.,
Barthel, G., Schuppler, J. and Schulte –
Hermann, R. (1984). Controlled Death
(apoptosis) of normal and putative
preneoplastic cells in rat liver following
withdrawal of tumor promoters.
Carcinogenesis, 5: 453-458.
Carthew, P., Edwards, R.E and Nolan, B.M.
(1998). The quantitative distinction of
hyperplasia from hypertrophy in
hepatomegaly induced in the rat liver by
phenobarbital. Toxicol. Sci., 44: 46 -
51.
Crawley, J., Waruiru, C., Mithwani, S. (2000).
Effect of Phenobarbital on seizure
frequency and mortality in childhood
cerebral malaria: a randomized,
controlled intervention Study. Lancet,
355: 701 – 706.
Crawley, J., Smith, S., Kirkham, F., Muthinji, P.,
Waruiru, C., and Marsh, K. (1996).
Seizures and status epilepticus in
childhood cerebral malaria. Q. J. Med.,
89: 591 - 597.
Garcia-Allan, C., Lord, P. G., Loughlin, J. M.,
Orton, T. C and Sidaway, J. E. (2000).
Identification of phenobarbitone-
modulated genes in mouse liver by
differential display. J. Biochem. Mol.
Toxicol., 14: 65 - 72.
Kokwaro, G. O., Ogutu, B. R., Muchohi, S. N.,
Otieno, G. O. and Newton, C. R. J. C.
(2003). Pharmacokinetics and Clinical
effect of Phenobarbital in children with
severe falciparum malaria and
convulsions. B., J. Clin. Pharmacol., 56
(4): 453 - 457
Lewin, E. and Bleck, V. (1997). Cyclic AMP
accumulation in cerebral slices: Effects
of carbamazepine, Phenobarbital and
phenytoin. Epilepsia, 18 (2): 237 - 242.
 Effects of phenobarbital administration on organ histology and biochemical activities of rats
Lopez – Munoz, F., Ucha-Udabe, R. and Alamo,
C. (2005). The history of barbiturates a
century after their clinical introduction.
Neuropsychiatry. Dis. Treat., 1 (4): 329 -
343.
Matsumoto, T., Hiramatsu, M. and Mori, A.
(1983). Effects of Phenobarbital on
serotonin level in forebrain and
cerebellum of mice with a hereditary
susceptibility to seizures. Biochem., 11
(9): 837.
Mayne, P. D. (1994). Clinical Chemistry in
diagnosis and treatment, 6th ed., Edward
Arnold, London.
Ogutu, B. R., Newton, C. R. J. C., and Crawley, J.
(2002). Pharmacokinetics and
anticonvulsant effects of diazepam in
children with severe falciparum malaria
and convulsions. Br. J. Clin. Pharmacol.,
53: 49 - 57.
View publication stats
450
st
Pal, D. K. (2003). Epilepsy control in the 21
century: leave no child behind.
Epilepsia, 44: 273 - 275.
Pal, D. K. (2007), Phenobarbital for Childhood
epilepsy: Systematic Review. Paediatr.
Perinat. Drug Ther., 7(1): 31-42.
Ruch, R. J. and Klaunig, J. E. (2003). Kinetics of
Phenobarbital inhibition of intercellular
communication in mouse hepatocytes.
Cancer Res., 48 (90): 2579 - 2522.
Shorvon, S. D. (1986) Drugs in developing
countries B. M. J., 292: 1666 -1667.
Shorvon, S. D and Farmer, P. J. (1988). Epilepsy
in developing Countries: a Review of
epidermiological, sociocultural and
treatment aspects. Epilesia, 29: 36-54.
Yazar, E. O., Demir, M. and Elmas (2002).
Phenobarbital effects on brain and liver
tissue enzyme activity in mice. Acta Vet.
Born, 71: 309 - 312.